Kajsa Paulsson

Tapasin is required for efficient peptide binding to transporter associated with antigen processing.

The transporter associated with antigen processing (TAP) binds peptides in its cytosolic part and subsequently translocates the peptides into the lumen of the endoplasmic reticulum (ER), where assembly of major histocompatibility complex (MHC) class I and peptide takes place. Tapasin is a subunit of the TAP complex and binds both to TAP1 and MHC class I. In the absence of tapasin, the assembly of MHC class I in the ER is impaired, and the surface expression is reduced. To clarify the function of tapasin in the processing of antigenic peptides, we studied the interaction of peptide and TAP, peptide transport across the membrane of the ER, and association of peptides with MHC class I molecules in the microsomes derived from tapasin mutant cell line 721.220, its sister cell line 721.221 expressing tapasin, and their HLA-A2 transfectants. The binding of peptides to TAP in tapasin mutant 721.220 cells was significantly diminished in comparison with 721.221 cells. Impaired peptide-TAP interaction resulted in a defective peptide transport in tapasin mutant 721.220 cells. Interestingly, despite the diminished peptide binding to TAP, the transport rate of TAP-associated peptides was not significantly altered in 721.220 cells. After transfection of tapasin cDNA into 721.220 cells, efficient peptide-TAP interaction was restored. Thus, we conclude that tapasin is required for efficient peptide-TAP interaction.

Chaperones and folding of MHC class I molecules in the endoplasmic reticulum.

In this review we discuss the influence of chaperones on the general phenomena of folding as well as on the specific folding of an individual protein, MHC class I. MHC class I maturation is a highly sophisticated process in which the folding machinery of the endoplasmic reticulum (ER) is heavily involved. Understanding the MHC class I maturation per se is important since peptides loaded onto MHC class I molecules are the base for antigen presentation generating immune responses against virus, intracellular bacteria as well as tumours. This review discusses the early stages of MHC class I maturation regarding BiP and calnexin association, and differences in MHC class I heavy chain (HC) interaction with calnexin and calreticulin are highlighted. Late stage MHC class I maturation with focus on the dedicated chaperone tapasin is also discussed.

Peptide-bound Major Histocompatibility Complex Class I Molecules Associate with Tapasin before Dissociation from Transporter Associated with Antigen Processing

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum.

Quality control of MHC class I maturation

Assembly of MHC class I molecules in the ER is regulated by the so‐called loading complex (LC). This multiprotein complex is of definite impor‐ tance for class I maturation, but its exact organization and order of assembly are not known. Evidence implies that the quality of peptides loaded onto class I molecules is controlled at multiple stages during MHC class I assembly. We recently found that tapasin, an important component of the LC, interacts with COPI‐coated vesicles. Biochemical studies suggested that the tapasin–COPI interaction regulates the retrograde transport of immature MHC class I molecules from the Golgi network back to the ER. Also other findings now propose that in addition to the peptide‐loading control, the quality control of MHC class I antigen presentation includes the restriction of export of suboptimally loaded MHC class I molecules to the cell surface. In this review, we use recent studies of tapasin to examine the efficiency of TAP, the LC constitution, ER quality control of class I assembly, and peptide optimization. The concepts of MHC class I recycling and ER retention are also discussed.—Paulsson, K. M., Wang, P. Quality control of MHC class I maturation. FASEB J. 18, 31–38 (2004)

The double lysine motif of tapasin is a retrieval signal for retention of unstable MHC class I molecules in the endoplasmic reticulum

Tapasin (tpn), an essential component of the MHC class I (MHC I) loading complex, has a canonical double lysine motif acting as a retrieval signal, which mediates retrograde transport of escaped endoplasmic reticulum (ER) proteins from the Golgi back to the ER. In this study, we mutated tpn with a substitution of the double lysine motif to double alanine (GFP-tpn-aa). This mutation abolished interaction with the coatomer protein complex I coatomer and resulted in accumulation of GFP-tpn-aa in the Golgi compartment, suggesting that the double lysine is important for the retrograde transport of tpn from late secretory compartments to the ER. In association with the increased Golgi distribution, the amount of MHC I exported from the ER to the surface was increased in 721.220 cells transfected with GFP-tpn-aa. However, the expressed MHC I were less stable and had increased turnover rate. Our results suggest that tpn with intact double lysine retrieval signal regulates retrograde transport of unstable MHC I molecules from the Golgi back to the ER to control the quality of MHC I Ag presentation.

Evolutionary and functional perspectives of the major histocompatibility complex class I antigen-processing machinery.

Abstract.Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8+ T cells, providing the basis for immune recognition of pathogen-infected cells. Peptides generated mainly by proteasomes in the cytosol are transported into the lumen of the endoplasmic reticulum by transporters associated with antigen processing (TAP). The maturation of MHC class I molecules is controlled by a number of accessory proteins and chaperones that are to a varying degree dedicated to the assembly of MHC class I. Several newly characterised proteins have been demonstrated to play important roles in this process. This review focuses on the functional relationship and evolutionary history of the antigen-processing machinery (APM) components and MHC class I itself. These are of great interest for further elucidating the origin of the immune system and understanding the mechanisms of antigen presentation and immunology in general.

Viral Proteins Interfering with Antigen Presentation Target the Major Histocompatibility Complex Class I Peptide-Loading Complex

The adaptive immune system is responsible for the final clearance and long lasting immunological memory of invading pathogens. Major histocompatibility complex class I (MHC-I) molecules play a central role in this defense as reporters of cellular content by presenting peptides derived from interior proteins in the cell. When recirculating cytotoxic T lymphocytes recognize MHC-I-presented peptides as foreign (e.g., derived from viral proteins), the presenting cell is killed by cytotoxic T lymphocytes, thereby hindering the spread of the virus. Thus, the key to efficient viral clearance by cytotoxic T lymphocytes lies within both the quality of the T-cell-receptor repertoire and the efficient processing and presentation of MHC-I-bound peptides. From the initial synthesis and folding to the final presentation on the cell surface, the MHC-I molecule gradually matures through multiple steps, most of which take place inside the endoplasmic reticulum (ER). The final stage of maturation for most MHC-I molecules takes place in the peptide-loading complex. The immune system and pathogens have evolved side by side for millions of years, and invading pathogens have developed several escape mechanisms to cripple the immune system. Among them are viral proteins that interfere with antigen presentation (VIPRs), which target both MHC-I and MHC-II antigen processing in order to skew or totally inhibit a functional immune response toward the virus. In this review, we discuss the main discoveries and latest developments concerning VIPRs that target the MHC-I peptide-loading complex.

The outermost N-terminal region of tapasin facilitates folding of major histocompatibility complex class I

Tapasin (Tpn) is an ER chaperone that is uniquely dedicated to MHC‐I biosynthesis. It binds MHC‐I molecules, integrates them into peptide‐loading complexes, and exerts quality control of the bound peptides; only when an “optimal peptide” is bound will the MHC‐I be released and exported to the cell surface for presentation to T cells. The exact mechanisms of Tpn quality control and the criteria for being an optimal peptide are still unknown. Here, we have generated a recombinant fragment of human Tpn, Tpn1–87 (representing the 87 N‐terminal and ER‐luminal amino acids of the mature Tpn protein). Using a biochemical peptide–MHC‐I‐binding assay, recombinant Tpn1–87 was found to specifically facilitate peptide‐dependent folding of HLA‐A*0201. Furthermore, we used Tpn1–87 to generate a monoclonal antibody, αTpn1–87/80, specific for natural human Tpn and capable of cellular staining of ER localized Tpn. Using overlapping peptides, the epitope of αTpn1–87/80 was located to Tpn40–44, which maps to a surface‐exposed loop on the Tpn structure. Together, these results demonstrate that the N‐terminal region of Tpn can be recombinantly expressed and adopt a structure, which at least partially resembles that of WT Tpn, and that this region of Tpn features chaperone activity facilitating peptide binding of MHC‐I.

Tapasin Facilitation of Natural HLA-A and -B Allomorphs Is Strongly Influenced by Peptide Length, Depends on Stability, and Separates Closely Related Allomorphs

Despite an abundance of peptides inside a cell, only a small fraction is ultimately presented by HLA-I on the cell surface. The presented peptides have HLA-I allomorph-specific motifs and are restricted in length. So far, detailed length studies have been limited to few allomorphs. Peptide–HLA-I (pHLA-I) complexes of different allomorphs are qualitatively and quantitatively influenced by tapasin to different degrees, but again, its effect has only been investigated for a small number of HLA-I allomorphs. Although both peptide length and tapasin dependence are known to be important for HLA-I peptide presentation, the relationship between them has never been studied. In this study, we used random peptide libraries from 7- to 13-mers and studied binding in the presence and absence of a recombinant truncated form of tapasin. The data show that HLA-I allomorphs are differentially affected by tapasin, different lengths of peptides generated different amounts of pHLA-I complexes, and HLA-A allomorphs are generally less restricted than HLA-B allomorphs to peptides of the classical length of 8–10 aa. We also demonstrate that tapasin facilitation varies for different peptide lengths, and that the correlation between high degree of tapasin facilitation and low stability is valid for different random peptide mixes of specific lengths. In conclusion, these data show that tapasin has specificity for the combination of peptide length and HLA-I allomorph, and suggest that tapasin promotes formation of pHLA-I complexes with high on and off rates, an important intermediary step in the HLA-I maturation process.

Tapasin Discriminates Peptide-Human Leukocyte Antigen-A*02:01 Complexes Formed with Natural Ligands

A plethora of peptides are generated intracellularly, and most peptide-human leukocyte antigen (HLA)-I interactions are of a transient, unproductive nature. Without a quality control mechanism, the HLA-I system would be stressed by futile attempts to present peptides not sufficient for the stable peptide-HLA-I complex formation required for long term presentation. Tapasin is thought to be central to this essential quality control, but the underlying mechanisms remain unknown. Here, we report that the N-terminal region of tapasin, Tpn1–87, assisted folding of peptide-HLA-A*02:01 complexes according to the identity of the peptide. The facilitation was also specific for the identity of the HLA-I heavy chain, where it correlated to established tapasin dependence hierarchies. Two large sets of HLA-A*02:01 binding peptides, one extracted from natural HLA-I ligands from the SYFPEITHI database and one consisting of medium to high affinity non-SYFPEITHI ligands, were studied in the context of HLA-A*02:01 binding and stability. We show that the SYFPEITHI peptides induced more stable HLA-A*02:01 molecules than the other ligands, although affinities were similar. Remarkably, Tpn1–87 could functionally discriminate the selected SYFPEITHI peptides from the other peptide binders with high sensitivity and specificity. We suggest that this HLA-I- and peptide-specific function, together with the functions exerted by the more C-terminal parts of tapasin, are major features of tapasin-mediated HLA-I quality control. These findings are important for understanding the biogenesis of HLA-I molecules, the selection of presented T-cell epitopes, and the identification of immunogenic targets in both basic research and vaccine design.